The physical and chemical factors that allow polynucleotides to perform their functions in the cell have been studied for several decades. Recent advances in the synthesis and manipulation of polynucleotides have allowed this field to move ahead especially rapidly during the past fifteen years. One of the most common chemical approaches to the study of interactions involving has been the use of nucleoside base analogs in which functional groups are added, deleted, blocked, or rearranged.
Such nucleoside analogs may be useful as in providing specific alterations to reaction kinetics; properties to oligonucleotide probes for diagnostic applications; to alter the properties of antisense RNA and RNAi; and in the synthesis and purification of oligonucleotides. Nucleoside analogs may also find use as metabolic inhibitors of viruses and proliferating cells, including tumor cells. Currently a number of nucleoside based drugs are being used to treat human diseases, including AIDS, against various cancers and for various systemic diseases resulting from inappropriate immune responses.
Among the uses of oligonucleotides are methods of inhibiting gene expression with antisense oligonucleotides complementary to a specific target messenger RNA (mRNA) sequences. Oligonucleotides also have found use in diagnostic tests performed using biological fluids, tissues, intact cells or isolated cellular components. For diagnostics, oligonucleotides and oligonucleotide analogs can be used in cell free systems, in vitro, ex vivo or in vivo.
Oligonucleotides and nucleosides are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of such other biological molecules. For example, oligonucleotides serve as primers in the reactions associated with polymerase chain reaction (PCR), which reactions are now widely used in forensics, paleontology, evolutionary studies and genetic counseling, to name just a few.
Nucleoside analogues that lack specific hydrogen bonding groups have proven useful in a number of biological contexts for probing the physical and chemical importance of such electrostatically charged moieties. For example, Strazewski and Tamm reported over two decades ago the synthesis of pyrimidine analogues lacking one of three hydrogen bonding groups, and investigated their substrate abilities with DNA polymerase enzymes. In another example, McLaughlin reported nucleobases with single functional groups deleted, and described their properties in pairing in DNA. Taking this approach to its logical limit, all Watson-Crick hydrogen bonding groups were removed in nucleoside analogues, preparing several “nonpolar nucleoside isosteres”, which maintain the steric size and shapes of natural nucleobases but lack polar functionality (Schweitzer and Kool (1994) J. Org. Chem. 59, 7238; Schweitzer and Kool (1995) J. Am. Chem. Soc. 117, 1863; Kool et al. (2000) Angew. Chem. Int. Ed. 39, 990. Examples included 4-methyl-aza-benzimidazole, an adenine mimic, and 2,4-difluorotoluene, a thymine mimic.
Nonpolar nucleoside isosteres have proven useful in probing the recognition of DNA by other nucleic acids, and in studies of the physical origins of DNA curvature. Biophysical studies have shown that thymine and adenine isosteres destabilize DNAs in which they are substituted, unless they are in a terminal position, in which case they can be strongly stabilizing, due to their avid stacking with natural DNA bases. Structural studies have shown that, despite the destabilization when present at non-terminal locations, thymine and adenine mimics show essentially the same structures as the natural congeners.
Nonpolar nucleoside mimics have also been increasingly useful of late in the study of protein-DNA and enzyme-DNA recognition. Studies have been reported with purine and pyrimidine mimics in a number of DNA repair enzymes, including MutY (Guckian et al. (1998) Nature Structural biology 5, 954); fpg; (Francis et al. (2003) J. Am. Chem. Soc. 125, 16235), MutS and homologues (Schofield et al. (2001) J. Biol. Chem. 276, 45505; Drotschmann et al. (2001) J. Biol. Chem. 276, 46225; and in polypurine tract recognition by HIV reverse transcriptase (Rausch et al. (2003) Proc. Natl. Acad. Sci. USA 100, 11279). Those studies have shed light on the relative importance of hydrogen bonding and steric interactions to these enzymes' biochemical activities.
In addition to this, nonpolar nucleoside isosteres have proven broadly useful in the study of DNA replication by a wide variety of polymerase enzymes. Such nonpolar analogues were first reported in 1997 to act as surprisingly strong substrates for DNA polymerase I, (Moran et al. (1997) J. Am. Chem. Soc. 119, 2056; Moran et al. (1997) Proc. Natl. Acad. Sci. USA 94, 10506) leading to the conclusion that at least some replicative DNA polymerases function well in synthesis of a base pair without Watson-Crick hydrogen bonds. This has since been confirmed by a number of studies of varied polymerase enzymes in vitro, and recently in living bacterial cells as well. The discovery of the lack of a hydrogen bonding requirement in replication has led to design other non-isosteric DNA base pairs for expansion of the genetic information-encoding system.
A high fidelity for DNA replication is required to maintain proper transfer of genetic information during cell division. The first and most influential step that determines this fidelity is synthesis of a new base pair by a replicative DNA polymerase. This choice, which occurs dozens of times per second, involves the selection of one nucleotide among four for insertion into the growing primer strand, opposite each DNA template base as it is addressed in turn. In eukaryotes, the replicative enzymes are DNA polymerases delta, alpha, and epsilon. In eubacteria, the replicative polymerases are Pol III, which synthesizes the leading strand, and Pol I, which assists Pol III with the lagging strand. These latter polymerases make an error (synthesis of a mismatched pair) only once in ca. 104-105 nucleotide insertions.
The biophysical origin of this fidelity is a long-standing topic of research on polymerases. Early studies often focused on matching of Watson-Crick hydrogen bonds; however, it was subsequently recognized that at the terminus of DNA, base pairing selectivity in the absence of enzymes is too low to account for the observed enzymatic fidelity. More recently, it has been shown that a nonpolar isostere of thymine (difluorotoluene) can be replicated with nearly wild-type fidelity despite its lack of hydrogen bonding ability (Moran et al. (1997) J. Am. Chem. Soc. 119, 2056-2057; Moran et al. (1997) Proc. Natl. Acad. Sci. USA 94, 10506-10511; Delaney et al. (2003) Proc. Natl. Acad. Sci. USA 100, 4469-4473). Such observations, in conjunction with structural and mutational studies, have led to the hypothesis that geometry of DNA base pairs may be regulated by a close fit in polymerase active sites (Kool (2002) Ann. Rev. Biochem. 71, 191-219; Kool (2001) Annu. Rev. Biophys. Biomol. Struct. 30, 1-33; Goodman (1997) Proc. Natl. Acad. Sci. USA 94, 4469-4473).
The development of novel nucleoside analogs is of interest for a variety of research and therapeutic uses. The present invention addresses this issue.